Use of supermeres derived from human umbilical cord mesenchymal stem cells in preparation of drugs for treating diabetic retinal fibrosis
Drugs prepared using Supermeres derived from human umbilical cord mesenchymal stem cells have overcome the shortcomings of existing technologies in inhibiting diabetic retinopathy, achieving significant effects in inhibiting fibrosis and promoting cell proliferation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU UNIV
- Filing Date
- 2025-12-11
- Publication Date
- 2026-06-05
AI Technical Summary
There is a lack of effective drugs to inhibit diabetic retinopathy in the current technology, and existing extracellular vesicle (EV) treatments have limited efficacy and insufficient targeting.
Supermeres derived from human umbilical cord mesenchymal stem cells were prepared using gradient ultracentrifugation to develop a drug for treating diabetic retinopathy. The drug significantly inhibited retinal cell fibrosis, reduced the expression of fibrosis marker proteins, promoted cell proliferation, and inhibited apoptosis.
Supermeres can significantly inhibit retinal cell fibrosis, reduce the expression of fibrosis marker proteins, significantly inhibit cell apoptosis, promote cell proliferation, and provide better therapeutic effects.
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Figure CN122140760A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Supermeres derived from human umbilical cord mesenchymal stem cells in the preparation of drugs for treating diabetic retinopathy. Background Technology
[0002] Globally, approximately 537 million adults are affected by diabetes, a number projected to reach 783 million by 2045. Of these, about 30% suffer from diabetic retinopathy (DR), currently affecting over 100 million adults worldwide, a number expected to increase to 160 million by 2045. Retinal fibrosis is a common pathological manifestation in the later stages of DR, characterized by excessive extracellular matrix (ECM) deposition, a significant cause of retinal detachment and blindness. Despite the use of new-generation drugs and vitreoretinal microsurgery in the clinical treatment of diabetic retinopathy, its prevalence continues to rise rapidly, and treatment outcomes for diabetic retinopathy fibrosis remain unsatisfactory. Therefore, the search for new and effective drugs to prevent and treat diabetic retinopathy and inhibit the progression of diabetic retinopathy fibrosis is an urgent clinical need.
[0003] Extracellular vesicles (EVs) are highly heterogeneous nanoparticles secreted by cells. Almost all cells can secrete EVs, which are widely distributed in cell culture supernatants and various body fluids, such as blood, urine, tears, saliva, amniotic fluid, and breast milk. EVs possess a phospholipid bilayer molecular membrane structure, enabling them to carry and deliver various bioactive substances, including proteins, lipids, nucleic acids, and metabolites. They can mediate cell proliferation, apoptosis, angiogenesis, and inflammation-related processes, thereby accelerating tissue repair and regeneration. However, due to the instability and insufficient targeting of EVs, their therapeutic effects are limited.
[0004] Supermeres are novel functional extracellular membrane-free nanoparticles discovered in the supernatant of extracted EVs. Compared to EVs, they exhibit greater uptake in vivo and are rich in bioactive substances associated with various cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1, and AGO2), Alzheimer's disease (APP), and cardiovascular diseases (ACE2, ACE, and PCSK9). Currently, there is no literature reporting whether Supermeres have therapeutic potential for diabetic retinopathy. Summary of the Invention
[0005] To address some shortcomings in existing technologies, this invention provides the application of human umbilical cord mesenchymal stem cell-derived Supermeres in the preparation of drugs for treating diabetic retinopathy. This invention has found that human umbilical cord mesenchymal stem cell-derived Supermeres can significantly inhibit retinal cell fibrosis and reduce the expression of fibrosis marker proteins α-SMA, col-1a1, and Fibronectin. Compared with human umbilical cord mesenchymal stem cell-derived EVs, it not only better inhibits the occurrence of fibrosis but also more significantly inhibits apoptosis and promotes cell proliferation, thereby achieving the technical effect of treating retinal fibrosis. The human umbilical cord mesenchymal stem cell-derived Supermeres have excellent applications in the preparation of drugs for treating diabetic retinopathy.
[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:
[0007] This invention first provides the application of human umbilical cord mesenchymal stem cells derived from Supermeres in the preparation of drugs for treating diabetic retinal fibrosis.
[0008] Preferably, the method for preparing the Supermeres includes:
[0009] Human umbilical cord mesenchymal stem cells were isolated and cultured, and the cells were expanded. After expansion, the cell supernatant was collected, and the cells were subjected to gradient ultracentrifugation to remove the clear liquid in the supernatant. The cells were then filtered to obtain the Supermeres.
[0010] Preferably, the gradient ultracentrifugation step includes:
[0011] Centrifuge at 300 g for 10 min, 2000 g for 10 min, 10000 g for 30 min, and 100000 g for 3 h. Collect the supernatant and centrifuge at 160000 g for 16 h. Collect the supernatant and centrifuge at 360000 g for 16 h. Discard the transparent liquid in the supernatant. After the lower red liquid is fully dissolved, filter it through a 0.22 μm sterile filter and store it at -80℃ for later use.
[0012] Preferably, the diabetic retinopathy includes diabetic retinopathy caused by retinal collagen deposition and retinal epithelial-mesenchymal transformation.
[0013] Preferably, the application includes protecting the integrity of the retinal structure and / or increasing retinal thickness.
[0014] Preferably, the application is as follows:
[0015] (1) Reduce retinal collagen deposition; (2) Inhibit the expression of one or more of the retinal cell fibrosis-related proteins COL1A1, Fibronectin and α SMA;
[0016] (3) Inhibit the expression of N-cadherin, Snail and vimentin, which are related to epithelial-mesenchymal transition;
[0017] (4) Promotes the expression of E-cadherin, a marker in epithelial cells;
[0018] (5) Inhibits apoptosis of retinal pigment epithelial cells;
[0019] (6) Promotes the proliferation of retinal pigment epithelial cells.
[0020] The present invention also provides a pharmaceutical composition for treating diabetic retinopathy, the pharmaceutical composition comprising Supermeres derived from human umbilical cord mesenchymal stem cells.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] This invention provides the application of human umbilical cord mesenchymal stem cell-derived Supermeres in the preparation of drugs for diabetic retinal fibrosis. The human umbilical cord mesenchymal stem cell-derived Supermeres described in this invention can significantly inhibit retinal cell fibrosis and reduce the expression of fibrosis marker proteins α-SMA, col-1a1, and Fibronectin. Compared with human umbilical cord mesenchymal stem cell-derived EVs, it can not only better inhibit the occurrence of fibrosis, but also more significantly inhibit cell apoptosis and promote cell proliferation, thereby achieving the technical effect of treating retinal fibrosis. Attached Figure Description
[0023] Figure 1 To observe the morphology of hucMSC-EVs and hucMSC-Supermeres using transmission electron microscopy.
[0024] Figure 2 The particle size and distribution of hucMSC-Evs (A) and hucMSC-Supermeres (B) were analyzed for nanoparticle tracking.
[0025] Figure 3Figure 1 shows the results of Western blotting detection of surface markers in hucMSC-EVs and hucMSC-Supermeres. Figure A shows the positive surface marker proteins ALIX, CD9, CD81, and TSG101 of hucMSC-EVs, and the negative surface marker protein Calnexin; Figure B shows the positive surface marker AGO2 and the negative surface marker Calnexin of hucMSC-Supermeres.
[0026] Figure 4 This is a schematic diagram illustrating the intervention of hucMSC-EVs and Supermeres in db / db mice to induce fibrosis.
[0027] Figure 5 Figure 1 shows the morphology and thickness analysis of the retinal tissue of db / db mice after H&E staining; A shows the HE staining results of the retina of db / m mice; B shows the HE staining results of the retina of db / db mice; C shows the HE staining results of the retina of db / db mice after hucMSC-EVs intervention; D shows the HE staining results of the retina of db / db mice after hucMSC-Supermeres intervention.
[0028] Figure 6 The image shows the results of Masson staining to observe collagen fiber deposition in the retinal tissue of db / db mice.
[0029] Figure 7 Figure showing the uptake of hucMSC-EVs (A) and hucMSC-Supermeres (B) by ARPE-19 cells.
[0030] Figure 8 The image shows the fibrosis status in ARPE-19 cells detected by Western Blot. In the image, A represents the expression of fibrosis proteins COL-1A1, Fibronectin, and α-SMA; B represents the statistical analysis results of the WB bands.
[0031] Figure 9 Figure showing the expression of fibrotic proteins α-SMA (A), COL1A1 (B), and Fibronectin (C) in ARPE-19 cells by immunofluorescence staining.
[0032] Figure 10 Western blotting was used to detect the epithelial-mesenchymal transition process in ARPE-19 cells, including the expression of N-Cadherin, E-Cadherin, Vimentin, and snail.
[0033] Figure 11Immunofluorescence staining was used to observe the epithelial-mesenchymal transition process of ARPE-19 cells; Figure A shows the expression of N-Cadherin observed by immunofluorescence staining; Figure B shows the expression of Vimentin observed by immunofluorescence staining.
[0034] Figure 12 The figure shows the statistical analysis results of ARPE-19 cell apoptosis rate detected by flow cytometry.
[0035] Figure 13 To detect the proliferation of ARPE-19 cells using CCK8 assay. Detailed Implementation
[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0037] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0038] Before obtaining Supermeres derived from human umbilical cord mesenchymal stem cells, this invention selects healthy maternal umbilical cords and obtains mesenchymal stem cells through isolation and culture. The supernatant of the cultured umbilical cord mesenchymal stem cells is then extracted using a gradient ultracentrifugation method.
[0039] The umbilical cord-derived mesenchymal stem cells described in this invention were isolated and cultured using existing isolation and culture methods (Qiao et al. Human mesenchymal stem cells isolated from the umbilical cord. Cell Biol Int. 2008 Jan;32(1):8-15).
[0040] The gradient ultracentrifugation method for obtaining hucMSC-EVs and Supermeres in this invention includes the following steps: centrifugation at 300 g for 10 min, centrifugation at 2000 g for 10 min, centrifugation at 10000 g for 30 min, centrifugation at 100000 g for 3 h, collecting the supernatant and centrifuging at 160000 g for 16 h, collecting the supernatant and centrifuging at 360000 g for 16 h, discarding the transparent liquid in the supernatant, and filtering the lower red liquid after it has been fully dissolved using a 0.22 μm sterile filter and storing at -80℃ for later use.
[0041] The EVs used in this invention were also obtained by ultracentrifugation. After centrifugation at 100,000 g for 3 h, the EVs were washed with PBS buffer and centrifuged at 100,000 g for 3 h to obtain the corresponding EVs precipitate. The precipitate was resuspended in the PBS buffer, filtered through a 0.22 μm sterile filter, and stored at -80°C for later use.
[0042] The present invention preferably uses a db / db mouse diabetic retinopathy model to verify the application effect of Supermeres derived from human umbilical cord mesenchymal stem cells.
[0043] Example 1: Preparation and Identification of Supermeres and EVs Derived from Human Umbilical Cord Mesenchymal Stem Cells
[0044] Fresh umbilical cord specimens were obtained from the Fourth Affiliated Hospital of Jiangsu University (Zhenjiang Maternal and Child Health Hospital) and approved by the Ethics Committee of Zhenjiang Maternal and Child Health Hospital (Ethics Review No. 201701). Primary human umbilical cord mesenchymal stem cells (hucMSCs) were successfully isolated using the umbilical cord tissue block adherence method. Supermeres and EVs derived from human umbilical cord mesenchymal stem cells were then prepared and identified. The materials used and their sources are shown below:
[0045] α-MEM culture medium (C12571500BT, Gibco), fetal bovine serum (F101-03, Vazyme), adipogenic and osteogenic induction reagent (HUXUC-90021, Cyagen Oricell), MSC surface labeling identification reagent (Cyagen Biosciences), RIPA lysis buffer + protease and phosphatase inhibitor mixed mini (Thermo Scientific™ + Pierce), CD9 (D8O1A) Rabbit mAb (13174S, CST), CD81 Polyclonal antibody (27855-1-AP, Proteintech), Alix (E6P9B) Rabbit mAb (92880S, CST), TSG101 Polyclonal antibody (28283-1-AP, Proteintech), Calnexin (C5C9) Rabbit mAb (2679S, CST), AGO2 antibody (67934-1-Ig, Proteintech)
[0046] The preparation and identification steps of the human umbilical cord mesenchymal stem cell-derived Supermeres and EVs are as follows:
[0047] S1. Isolation, culture, and identification of HucMSCs:
[0048] After removing the umbilical veins and arteries from the human umbilical cord tissue, it is cut into 1mm pieces. 3 Small pieces are placed in a culture dish, and the culture medium containing 15% fetal bovine serum is replaced every two days. When enough cells have emerged, they are digested to obtain human umbilical cord mesenchymal stem cells.
[0049] The obtained human umbilical cord mesenchymal stem cells were cultured in α-MEM nutrient medium containing 10% fetal bovine serum at 37°C and 5% CO2 in a saturated humidity incubator. The culture results showed that hucMSCs exhibited spindle-shaped structures and fish-like colony growth. Figure 1 When hucMSCs reached the 2nd or 3rd generation, the cells were digested and seeded into six-well plates. Differentiation induction experiments were performed using osteogenic and adipogenic induction reagents, and their differentiation capacity was then observed using Alizarin Red staining and Oil Red O staining, respectively. The results showed that after induction, hucMSCs exhibited calcium nodules and oil droplets, indicating that the hucMSCs cultured in this example possessed the pluripotent differentiation characteristics of mesenchymal stem cells.
[0050] Flow cytometry surface marker identification of hucMSCs showed that CD105, CD73, and CD29 were positive, while CD45, CD14, and CD11b were negative, indicating that the hucMSCs cultured in this invention have the surface characteristics of mesenchymal stem cells.
[0051] S2. Preparation and identification of hucMSC-Supermeres and hucMSC-EVs:
[0052] The human umbilical cord mesenchymal stem cells obtained in step S1 with good growth status were seeded in a large culture dish and cultured in α-MEM complete medium containing 10% FBS until 40%-50% confluence. After washing twice with PBS, the culture medium was replaced with 8 mL / dish of exosome-free serum-conditioned medium and cultured for another 48 h. The supernatant was then collected and the cells were passaged at a ratio of 1:3. The above operation was repeated until passage P7.
[0053] hucMSC-Supermeres and hucMSC-EVs were prepared using a gradient ultracentrifugation method. The collected culture supernatant was sequentially centrifuged at 2000×g for 30 min at 4°C to remove dead cells, and then centrifuged at 10000×g for 30 min to remove cell debris. Pre-concentration was then performed using a 100 kDa ultrafiltration centrifuge tube (2000×g, 30 min). This step was repeated until a suitable volume of dark-colored concentrate was obtained. The concentrate was then transferred to an ultracentrifuge tube and centrifuged at 100000×g for 3 h to obtain the precipitate as hucMSC-EVs. After washing with PBS, the precipitate was purified by another centrifugation at 100000×g for 3 h. The precipitate was resuspended in an appropriate amount of PBS, aseptically filtered through a 0.22 μm filter, aliquoted, and stored at -80°C for later use.
[0054] Collect the supernatant after removing hucMSC-EVs, centrifuge at 160,000×g for 16 h to remove the precipitate, and continue to centrifuge the supernatant at 360,000×g for 16 h to obtain a viscous or gel-like precipitate, which is hucMSC-Supermeres. Resuspend in an appropriate amount of PBS, filter aseptically through 0.22 μm, aliquot and freeze at -80℃ for later use.
[0055] like Figure 1-3 As shown, the hucMSC-Supermeres obtained in this step are approximately 20-50 nm in size and lack membrane structure. The hucMSC-EVs exhibit a typical biconcave disk shape with an average particle size of approximately 150 nm. Western blot analysis revealed that hucMSC-Supermeres express the positive biomarker AGO2, while hucMSC-EVs express the surface positive biomarkers Alix, TSG101, CD9, and CD81, but do not express the negative biomarker Calnexin.
[0056] Example 2: Evaluation of hucMSC-Supermeres intervention
[0057] In this embodiment, the db / db mouse diabetic retinal fibrosis model was used to intervene in the hucMSC-Supermeres obtained in Example 1, and the intervention results were examined.
[0058] The main materials used in this embodiment and their sources are as follows:
[0059] Microsyringe (7634-01, Hamilton) and microsyringe needle (7803-05, Hamilton);
[0060] Male db / m mice (normal non-diabetic mice, 8 weeks old, approximately 24g, purchased from Jingweiyu Biotechnology Co., Ltd.);
[0061] Male db / db mice (spontaneously obese diabetic mice, 8 weeks old, approximately 40-55g, purchased from Jingweiyu Biotechnology Co., Ltd.);
[0062] The concentrations of hucMSC-EVs and hucMSC-Supermeres collected in Example 1 were adjusted to 1×10 using sterile PBS. 8 Particle number / μL.
[0063] The examination steps in this embodiment are as follows:
[0064] Male db / m mice were used as the control group, and male db / db mice were used as the experimental group. All animals were housed in an environment free of pathogens, with ample food and water, a temperature of 25°C, and a 12-hour light / dark cycle. Figure 4 The procedure shown below is used to obtain the experimental group and the control group. The specific steps are as follows:
[0065] Experimental group (db / db group): Male db / db mice were raised to 16 weeks of age to obtain a mouse model of retinal fibrosis. These db / db mice were then divided into 3 groups, and each group was injected with 1×10 8 1 μL of hucMSC-EVs and hucMSC-Supermeres were added to 1 μL of PBS, and the animals were fed to continue to be cultured for 20 weeks, followed by in vivo injection of 1 × 10⁻⁶ ppm. 8 One μL of hucMSC-EVs and hucMSC-Supermeres were added to one μL of PBS to obtain male db / db mice treated with hucMSC-EVs and hucMSC-Supermeres, as well as untreated db / db mice. These two groups of male db / db mice treated with hucMSC-EVs and hucMSC-Supermeres served as controls, and were observed in subsequent experiments.
[0066] Control group (db / m group): Male db / m mice were raised to 16 weeks of age. 1 μL of PBS was injected into the male db / m mice via the intravitreal cavity. The mice were raised to 20 weeks of age and then injected with 1 μL of PBS in vivo to obtain untreated male db / m control mice.
[0067] The control and experimental groups were fed until 24 weeks of age, then euthanized to harvest the eyeballs and assess the pathological changes in retinal tissue after intervention with hucMSC-EVs and hucMSC-Supermeres.
[0068] Retinal tissue from mice in each group was sectioned and stained with H&E. The retinal thickness at a distance of 400 μm from the optic nerve head was measured in each group. The average thickness was 220 μm in the d / m group, 160 μm in the db / db group, 195 μm in the hucMSC-EVs group, and 225 μm in the hucMSC-Supermeres group. Figure 5 As can be seen, compared with the db / m group, the retinal thickness of mice in the db / db group was significantly thinner. hucMSC-EVs and hucMSC-Supermeres can increase the thickness of the mouse retina and improve the structure of various layers, including the retinal ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Compared with hucMSC-EVs, hucMSC-Supermeres has a more significant effect on restoring retinal thickness. Figure 8 Masson staining showed that intervention with hucMSC-EVs and hucMSC-Supermeres effectively reduced collagen fiber deposition on the inner surface of the mouse retina, with hucMSC-Supermeres showing a more significant effect. Figure 6 ).
[0069] In summary, the hucMSC-Supermere of this invention can better improve the disordered structure of each layer of the retina in db / db mice, increase retinal thickness, reduce collagen fiber deposition, and inhibit the occurrence and development of fibrosis, providing a new strategy and experimental basis for the treatment of fibrosis in diabetic retinopathy.
[0070] Example 3: Investigation of the inhibitory effect of hucMSC-Supermeres on ARPE-19 cell fibrosis
[0071] The retina is composed of multiple layers of orderly arranged cells, interspersed with a rich network of blood vessels. In diabetic retinopathy, the pathological microenvironment leads to abnormal functional changes in various cells, among which ARPE-19 cells play a crucial role in the development of retinal fibrosis. Therefore, inhibiting the pathological changes in ARPE-19 cells is extremely important for the treatment of retinal fibrosis.
[0072] This embodiment examines the effect of hucMSC-Supermeres obtained in Example 1 on inhibiting ARPE-19 cell fibrosis, and the in vitro repair effect of hucMSC-Supermeres on retinal fibrosis.
[0073] The main materials used and their sources are shown below:
[0074] COL1A1 antibody (BS70155, Bioworld), α-Smooth Muscle Actin (ACTA2) antibody (A17910, Abclonal), Fibronectin antibody (15613-1-AP, Proteintech), D(+) anhydrous glucose powder (63005518, Sinopharm Group), DMEM low glucose culture medium (Bioind 01-051-1ACS);
[0075] ARPE-19 (i.e., human retinal pigment epithelial cells, purchased from the Cell Bank of the Chinese Academy of Sciences).
[0076] Dissolve D(+) anhydrous glucose powder in DMEM low-glucose culture medium to prepare a nutrient solution containing 30mM glucose, i.e., a high-glucose nutrient solution.
[0077] The examination steps in this embodiment are as follows:
[0078] ARPE-19 cells were cultured in low-glucose DMEM medium until confluence reached 80-90%, then digested with trypsin and seeded into 6-well plates. Cells were stimulated with high-glucose medium to simulate the high-glucose environment of diabetes; 1×10⁶ cells were added to each well of the intervention group. 8 ARPE-19 cells were obtained by treating hucMSC-Supermeres (with a higher particle count) and the control group (with the same dose of hucMSC-EVs) for 48 h. A portion of the treated ARPE-19 cells was used to extract cellular proteins for Western blotting, and the other portion was seeded onto cell slides for immunofluorescence assays. The results are shown below. Figures 7-9 As shown.
[0079] This embodiment demonstrates through a retinal cell uptake experiment that hucMSC-Supermeres can be taken up by ARPE-19 cells. Figure 10 By simulating a fibrotic environment with a high-glucose setting, ARPE-19 cells underwent fibrosis-like changes. Western blot results showed that compared to the low-glucose group, the expression of fibrosis-related proteins in ARPE-19 cells was significantly increased under a high-glucose environment, including Fibronectin, a major component of the extracellular matrix, COL1A1, and α-SMA, a marker of fibroblasts. Intervention with hucMSC-Supermeres and hucMSC-EVs inhibited this change, reducing the expression levels of Fibronectin, COL1A1, and α-SMA proteins after high-glucose stimulation. Among these, hucMSC-Supermeres showed better inhibition of fibrosis-related protein expression compared to hucMSC-EVs. Figure 8 Immunofluorescence staining showed that the levels of fibrosis-related proteins were elevated in ARPE-19 cells treated with high glucose, and these proteins accumulated in large quantities in the cytoplasm. Intervention with hucMSC-EVs and hucMSC-Supermeres inhibited the expression of Fibronectin, COL1A1, and α-SMA proteins in the cells, with hucMSC-Supermeres showing a more significant inhibitory effect. Figure 9 ).
[0080] Example 4: Investigation on the inhibition of ARPE-19 cell epithelial-mesenchymal transition by hucMSC-Supermeres
[0081] The main materials and their sources used in this embodiment are as follows:
[0082] E-cadherin antibody (BS1098, Bioworld), N-cadherin antibody (13116T, CST), Vimentin antibody (5741T, CST), Snail antibody (3895S, CST), Annexin V-FITC / PI apoptosis detection kit (A211-01, Vazyme), Transwell cell migration plate (3422, Corning), D(+) anhydrous glucose powder (63005518, Sinopharm Group), and crystal violet staining solution (G1062, Solarbio).
[0083] ARPE-19 (i.e., human retinal pigment epithelial cells, purchased from the Cell Bank of the Chinese Academy of Sciences).
[0084] Dissolve D(+) anhydrous glucose powder in DMEM low-glucose culture medium to prepare a nutrient solution containing 30mM glucose, i.e., a high-glucose nutrient solution.
[0085] The examination steps in this embodiment are as follows:
[0086] S1. Investigation on the inhibition of epithelial-mesenchymal transition in ARPE-19 cells by hucMSC-Supermeres:
[0087] In ARPE-19 cells, a high-glucose environment can induce epithelial-mesenchymal transition (EMT), causing cells to lose their original epithelial phenotype and transform into mesenchymal cells. EMT in ARPE-19 cells not only directly promotes retinal fibrosis but also impairs their physiological barrier function, allowing inflammatory mediators and other pathogens from within the blood vessels to enter the retina and damage other retinal cells, further exacerbating retinal disease. Therefore, this study further investigates whether hucMSC-Supermeres can better inhibit EMT in ARPE-19 cells, thereby better protecting retinal function and inhibiting the progression of retinal fibrosis. The specific steps are as follows:
[0088] ARPE-19 cells were cultured in low-glucose DMEM medium until confluence reached 80-90%, then digested with trypsin and seeded into 6-well plates. The cells were then stimulated with high-glucose medium to simulate the high-glucose environment of diabetes. In the intervention group, 1×10⁶ cells were added to each well. 8 The number of hucMSC-Supermeres was increased, while the control group was treated with the same dose of hucMSC-EVs for 48 h. After treatment, a portion of the cells were extracted for Western blotting experiments, and the other portion was seeded onto cell slides for immunofluorescence experiments.
[0089] Western blot results showed that, compared with the low glucose group, the expression of E-cadherin, an epithelial cell marker, was downregulated in ARPE-19 cells under high glucose conditions, while the expression of N-cadherin, Vimentin, and Snail, mesenchymal cell markers, was increased. Intervention with hucMSC-EVs and hucMSC-Supermeres increased the expression of the cell adhesion protein E-cadherin and reduced the expression of EMT-promoting proteins, effectively maintaining the epithelial characteristics of ARPE-19 cells. hucMSC-Supermeres, compared with hucMSC-EVs, was better able to maintain the epithelial characteristics of ARPE-19 cells. Figure 10 Immunofluorescence results further indicated that N-cadherin, a protein mediating cell adhesion, is mainly located on the ARPE-19 cell membrane. After high glucose treatment, E-cadherin, which maintains intact epithelial cell junctions, was replaced by N-cadherin. hucMSC-Supermeres further downregulated N-cadherin expression, thereby strengthening tight cell junctions and preventing cell polarity loss. In addition, the mesenchymal marker Vimentin in ARPE-19 cells was further downregulated after hucMSC-Supermeres intervention. Figure 11This indicates that hucMSC-Supermeres, compared to hucMSC-EVs, can better inhibit the epithelial-mesenchymal transition process of ARPE-19 cells and better inhibit the progression of retinal fibrosis.
[0090] An investigation into the inhibitory effect of S2.hucMSC-Supermeres on ARPE-19 cell apoptosis and promotion of ARPE-19 cell proliferation:
[0091] This step involved pretreating ARPE-19 cells using the EDU apoptosis kit, followed by flow cytometry analysis to detect apoptosis in ARPE-19 cells under different treatment conditions. The results are shown below. Figure 12 and Figure 13 As shown.
[0092] Combination Figure 12 and Figure 13 It can be seen that, compared with hucMSC-EVs, hucMSC-Supermeres can better inhibit the apoptosis level of ARPE-19 cells. Figure 12 Meanwhile, CCK8 proliferation experiments also found that hucMSC-Supermeres significantly promoted the proliferation level of ARPE-19 cells. Figure 13 This indicates that hucMSC-Supermeres can better promote ARPE-19 cell proliferation and inhibit apoptosis compared to hucMSC-EVs, and may play a better role in inhibiting retinal fibrosis.
[0093] In summary, this invention has found that human umbilical cord mesenchymal stem cell-derived Supermeres can significantly inhibit retinal cell fibrosis and reduce the expression of fibrosis marker proteins α-SMA, col-1a1, and Fibronectin. Compared with human umbilical cord mesenchymal stem cell-derived EVs, Supermeres not only better inhibit the occurrence of fibrosis but also more significantly inhibit apoptosis and promote cell proliferation, thereby achieving the technical effect of treating retinal fibrosis. The human umbilical cord mesenchymal stem cell-derived Supermeres have excellent applications in the preparation of drugs for treating diabetic retinopathy.
[0094] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.
Claims
1. Application of human umbilical cord mesenchymal stem cell-derived supermeres in the preparation of drugs for the treatment of diabetic retinopathy.
2. The application according to claim 1, characterized in that, The method for preparing the Supermeres includes: Human umbilical cord mesenchymal stem cells were isolated and cultured, and the cells were expanded. After expansion, the cell supernatant was collected, and the cells were subjected to gradient ultracentrifugation to remove the clear liquid in the supernatant. The cells were then filtered to obtain the Supermeres.
3. The application according to claim 2, characterized in that, The gradient ultracentrifugation steps include: Centrifuge at 300g for 10 min, 2000g for 10 min, 10000g for 30 min, and 100000g for 3 h. Collect the supernatant and centrifuge at 160000g for 16 h. Collect the supernatant and centrifuge at 360000g for 16 h. Discard the transparent liquid in the supernatant. After the lower red liquid is fully dissolved, filter it through a 0.22μm sterile filter and store it at -80℃ for later use.
4. The application according to claim 1, characterized in that, The diabetic retinopathy includes diabetic retinopathy caused by retinal collagen deposition and retinal epithelial-mesenchymal transformation.
5. The application according to claim 1, characterized in that, The applications include protecting the integrity of retinal structures and / or increasing retinal thickness.
6. The application according to claim 1, characterized in that, The application includes any of the following: (1) Reduce retinal collagen deposition; (2) Inhibit the expression of one or more of the retinal cell fibrosis-related proteins COL1A1, Fibronectin and αSMA; (3) Inhibit the expression of N-cadherin, Snail and vimentin, which are related to epithelial-mesenchymal transition; (4) Promotes the expression of E-cadherin, a marker of epithelial cells; (5) Inhibits apoptosis of retinal pigment epithelial cells; (6) Promotes the proliferation of retinal pigment epithelial cells.
7. A pharmaceutical composition for treating diabetic retinopathy, characterized in that, The pharmaceutical composition contains Supermeres derived from human umbilical cord mesenchymal stem cells.